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University of Nigeria Research Publications University of Nigeria Research Publications NNAMANI, Petra Obioma Author PG/M.PHARM/02/32723 Studies on Mucin Extracted from the Giant Snail Archachantina Marginata Title (Fam. Arionidea) Pharmaceutical Sciences Faculty Pharmaceutics Department June, 2004 Date Signature S'FUDIES ON MUCIN EXTH ACTED FROM THE GIANT SNAIL ARCHACHA TINA M,4 IIGIM TA, FAM. ARIONIDAE NNAMANI, PET1.U OBIOMA (PGIM. PHARM..102/32723) DEPARTMENT OF PHARMACEUTICS, FACULTY OF PHARMACEUTICAL SCIENCES, UNIVERSITY OF NIGERIA, NSUKKA JUNE, 2004 STUDIES ON MUCIN EXTR ' TEDFROM. THE GIANT SNAIL A RCHA CHA TINA MAH r;!lNATA, FAM. ARIONIDAE NNAMANI, PETRA ORIOMA (PG/M. : IARM./02/3 2723) A DISSERTATION SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF THE MASTER OF PHARMACY (M. PEIARM.) DEGREE OF THE UNIVERSITY OF NIGERIA, NSUKKA. SUPERVISOR: DR. M. U. ADIKWU JUNE, 2004 iii CER'I'I I (CATION Nnanlani, Petra Obioma, a postgraduate :tr~dent in the Department of Pharrnaceutics and with the Reg. No. PGIM. PHARM.10213272 ' 1 ;satisfactorily completed the requirements for research work for the degree of Master of 1 harmacy in Pllarmaceutics. The work embodied in this dissertation is original and has not bep 1 submitkd in part or full for any other diploma or degree of this or any other University. Dr. V.C. Okore (Head of Department) (Supervisor) "\x!.kr, \~WQ&2 %L? Date:. .. .. ... .. .. .. .. DEi 'ATION This work is dedicated to: M'Y ' ..'H'I LD i 'EN ; Eze-Nnamani, Cecil J. J. and Petra Ugochi~lyere~n,wl'hout whose companion this work would have been cc !pleted much ~arlier. M-.:!IUSBAND ; Dr. f t!:l.il Eze Nnamani, for his affection, inspira ' >n,understanding anti .;:.ern. MY' L'4RENTS; Mr. And Mrs. Gabriel Chukwuma Ukwucz . and Mr. And Mrs. Pius Ugwoke Nnamani for their mcral support. And, MY SIELINGS ; Engr. Bona, Sr. Ann, Mum. Doris, Jude, Felicia, Crescent, Vitalis, Lilian and Perpetua for their understant ling and concern. v ACKNOWI. ; BGEMENT I am greatly indebted to my supervie ,r. ,. M. U. Adikwu for his expert advice, encouragement, assistance and constructive r riticisnl3 that made the successful completion of this work possible. I am also greatly indebted to C\r. A. A. Attama for his invaluable assistance, encouragement, intelligent instructions, di~,%ctions and guidance throughout the period of the work and as always. I could never have iniv I: it without you. I also thank Dr. C. 0. Esimone for his advice and encouragement. May God b ess you all. My sincere thanks go to Dr. F.0.C Nwodo for his kind assistance and advice. I wish also to express my appreciation to Drs. Iherlioha J.I. for his wonderful assistance in the work and friendly sharing of ideas always. I also wish '0 appreciate Dr. Charles Okoli, Pharms. Ogbonna Okorie, Udeogaranya, Ezejiofor, Ofokansi, Mr. Uzuegbu Dave, Mrs. Ng Nwodo and Mrs. U. C. Odo for their assistance and friendly sharing of ideas during the period of the work and as always. I also immensely thank Prof. P. A. Akah and Dr. G. C. Onunkwo for their support and co-operation. My sincere gratitude also goes to Mr. Awa Uka, Mr. Ogboso and other staff of the Department of Pharmaceutics for their kind assistance in many areas of the work. I must as a matter of necessity acknowledge the warmth of some friends and colleagues Pharms. Nzekwe, Bzea, Ike-Uno, Onugu, Dieke, Nwozuzu, Izuegbu and Nnaeto. Finally, I thank God for my life, my family, well-wishers and more especially for the successful completion of this work. However, it is difficult to acknowledge everyone who lent a helping hand in one way or the other to see this research completed successfully. I only pray God to bless and reward all of them abundantly. Nnamani, P. 0. (Nee Ukwueze) TABLE OF ('ONTENTS Title Page ... ii ... Certification ... 111 Dedication ... iv Acknowledgement v Abstract xii Table of contents vi CHAPTER ONE 1.0 Introduction 1 I . I Bioadhesion 1 1.1.1 Mechanism of Bioadhesion ... 1 1.1.2 Forces of adhesion ... ... 3 1 . 1.3 Factors affecting bioadhesion ... 4 1.1.3.1 Mucin - mucin cohesive forces 4 1.1.3.2 Molecular weight of adhesive material 4 1.1.3.3 Coat weight ... ... ... ... 5 1.1 3.4 Hydrogen ion concentration ... ... 5 1.1.3.5 Hydration of adhesive material ... 5 1.1.3. G Presence of electrostatic charges ... G 1.1.4 The bioadhesive mucus ... ... 6 1 . 1.5 Adhesive polymers ... ... ... 7 1 I 0 Carboxyvinyl polymer (carbopolTM)... 9 I . I .0. I Carbomer (Carbopol Ultrez- I o'~)... 10 1.1.7 Sodium carboxymethylcellulose ... 11 1.1.8 Tragacanth ... ... ... , 12 1.1.9 Gelatin ... ... ... ..a 12 1.1.10 Methods of measuring bioadhesive strength of polymer 12 vii 1.1.1 1 Applications of bioadhesion ... ... ... ... ... ... ... 14 1.2 Dissolution ... .. ... ... ... ... ..a ... ... 15 1.2.1 Methods of studying dissolution ... ... ... ... ... ... 16 1.2.1.1 Modified USP disintegration apparat~ls ... ... ... ... ... 16 1.2.1.2 The rotating basket assembly ... ... ... ... ... ... ... 16 1.2.1.3 The paddle mixer ... ... ... ... ... ... ... ... 17 1.2.1.4 Rotary impellers ... ... ... ... .a. ... ... 18 1.2.2 Factors affecting dissolution rates of dnrgs in t.!e gastrointestinal tract ... 19 1.2.2.1 Physiological condition ... ... ... ... ... ... ... 19 1.2.2.2 Physicochemical properties ... ... ... ... ... ... ... 20 1.3 Gastrointestinal motility ... ... ... ... ... ... 23 1.4 Action on the peripheral nervous system ... ... ... ... ... 24 1.5 Medicament ... ... ... ... ... ... ... ... 25 1.5.1 Chlorpropamide ... ... ... ... ... ... ... ... 26 1.6 Viscosity. rheology and the flow of fluids ... ... ... ... ... 27 1.6.1 Newtonian fluids ... ... ... ... ... ... ... 28 1.6.1.1 Dynamic viscosity ... ... ... ... ... ... ... ... 28 1.6.2 Non-Newtonian fluids ... ... ... ... ... ... ... ... 31 1.6.2.1 Types of non-Newtonian behaviour ... ... ... ... ... ... 31 1.6.3 Methods used in determining the flow properties of simple fluid ... ... 33 1 h.4 Determination of the flow properties of non-Newtonian fluids ... ... 33 1.7 Gel permeation with Sephadex ... ... ... ... ... ... 34 1.7.1 Crosslinked dextran (Sephadex) properties ... ... ... ... ... 35 1.7.1.1 Physical properties ... ... ... ... ... ... ... ... 35 1.7.1.2 Chen~icalstability of Scphadcx ... ... ... ... ... ... 36 1.7.2 Applications of Sephadex ... ... ... ... ... ... ... 36 1.7.2.1 Analysis of multi-component samples ... ... ... ... ... 36 1.7.2.2 Determination of molecular weights ... ... ... ... ... ... 37 1.7.2.3 Zone electrophoresis ... ... ... ... ... ... ... ... 37 1.8 Mucins ... ... ... ... ... ... ... ... ... 38 1.8.1 The biological mucus membrane ... ... ... ... ... .... 39 viii 1.8.2 Cl~aracterizationof much ... ... ... ... ... ... 1.8.3 Interaction of mucin with biological con~pounds ... ... ... 1.8.4 A new glycosaminoglycan from the giant African snail Achafinafilica 1.9 Snails ... ... ... ... ... ... 1.9.1 The giant land snails of West Africa ... ... 1.9.2 The genus Ackatincr ... ... ... ... 1.9.3 Achatina (Achatina) achatina (Linnk) ... ... 1.9.4 Common species of the genus Archachatina ... 1.9.4.1 Archachatina (calachatina) inarginata (Swainson) 1.9.4.2 Archachatina (Calachatina) dcgreri Bequaert ... 1.9.4.3 Archachatina (calachatina) ventricosa (Gould) 1.10 Preservation of snails ... ... ... ... 1.1 1 'I'he Giant African snail: Achntinnficlica Fen~ssae 1.12 Physical requirement and production ... ... 1.13 'The control of Achntina fulica ... ... 1.13.1 Chemical control ... ... ... ... 1.13.2 Collection and destruction ... ... ... 1.13.3 Other control and protective measures ... 1.13.4 Biological control ... ... 1.14 In search of protein: Snailery ... 1.15 Analgesic potentials of snail ... 1.16 Objectives of the study ... CHAPTER TWO 2.0 MATERIALS AND METHODS 2.1 Materials ... ... ... 2.1.1 Snails ... ... ... ... 2.1.2 Animals ... ... ... 2.2 Methods ... ... ... 2.2.1 Extraction of snail mucin ... 2.2.2 Pliysicochemical tests ... ... , ... ... ... ... 2.2.2.1 Test for sugars ... ... ... ... ... ... ... ... 2.2.2.2 Test for carbol~ydrates(reduction test) ... ... ... ... 2.2.2.3 Test for proteins (amines; oxidation test) ... ... ... ... 2.2.2.4Test for fixed oils ... ... ... ... ... ... ... 2.2.2.5Solubility profile of the snail inucin ... ... ... ... ... 2.2.3 Preparation of snail rnucin dispersion ... ... ... ... ... 2.2.4 Rheological study on aqueous snail mwin dispersion ... ... 2.2.5 Type of flow ... ... ... a,. ... ... ... ... 2.2.6 Effect of concentration of the snail much ... ... ... ... 2.2.7 Effect of temperature ... ... ... ... ... ... ... 2.2.8 Effect of electrolytes ... ... ... ... ... ... ... 2.2.9 Effect of polymers ... ... ... ... , .. ... ... 2.2.10 Effect of aging ... ... ... ... ... ... ... ... 2.2.1 1 Molecular weight determination by gel permeation chromatography on Sephadex G-200 ... ... ... ... ... 2.2.12 Determination of snail mucin isoelectric point ... ... ... 2.2.13 Test for allergic properties ... ... ... .. ... ... 2.2.13.1 Preparation of stock solution ... ... ... ... ... 2.2.13.2Guinea pig wheal test ... ... ... ... ... ... 2.2.14Toxicological studies ... ... ... ... ... ... ... 2.2.14.1 LDSodeterminatioil ... ... ... ... ... ... ... 2.2.14.2Chronic toxicity determination ... ... ... ... ... 2.2.15 'I'ensiometric determination of bioadhesive strength of snail mucin ... 2.2.15.1 Preparation of snail mucin stock ... ... ... ... ... 2.2.15.2The bioadhesive strength ... ... ... ... ... ... 2.2.16 Evaluation of the bioadhesive strength of the snail mucin using coated glass beads ... ... ... ... ... 2.2.16.1 Preparation of simulated gastric fluid (SGF)
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